Uso de técnicas moleculares para determinação de Streptococcus pneumoniae e sorotipos colonizadores da nasofaringe na era pós-vacinal
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2013-01-23
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Universidade Federal de Goiás
Resumo
Brazil was the first country to introduce the pneumococcal conjugate 10valent
vaccine into the National Immunization Program for infants, in 2010. The
nasopharyngeal colonization by Streptococcus pneumoniae occurs early in life. It is the
first step for the development of invasive diseases. So far no study has evaluated the
impact of vaccination on the reduction on pneumococcal carriage. The evaluation of the
impact of vaccination should based on technologies with high accuracy. In this
investigation we applied molecular technologies, recently developed, to ascertain
pneumococcal nasopharyngeal colonization and serotypes. Objectives: (i) to compare
the prevalence of S. pneumoniae nasopharyngeal colonization by using real-time PCR
(RT-PCR) and multiplex PCR, and culture (“gold standard”) in children residing in
Goiania municipality; (ii) to evaluate the simultaneous colonization by different
serotypes by using the multiplex PCR technique. Methods: A household populationbased
survey was carried out between October/2010 and March/2011 by using a
systematic sampling, weighted by census tract. Based on previous studies, the sample
size was calculated taking into account an estimated 50% of pneumocococcal carriage.
A total of 1,437 nasopharyngeal swabs were collected from children less than 24
months of age. Broth-enriched culture of nasopharynx specimens followed by
pneumococcal isolation by both, culture and RT-PCR targeting the lytA gene (S.
pneumoniae) were performed. Pneumococcal carriage was defined for RT-PCR Cycle
threshold (Ct) < 35.0, and therefore all samples were submitted to multiplex PCR to
detect serotypes. ROC curve (Receiver Operating Characteristics) were built up to
identify Ct values predicted of S. pneumoniae positive culture. Results: The prevalence
of pneumococcal carriage by RT-PCR (56.9%) was statistically higher (p< 0,001),
compared to that obtained by culture (39.3%), regardless of the vaccination status.
Among the 818 positive children/samples by RT-PCR, in 54.2% of them it was possible
to detect the serotype. Simultaneous colonization by different types was found in 6.9%
of the children. Ct values Ct33.0 showed the best accuracy (91.4%) to predict positive
pneumococcal culture (Sensitivity=88% and Specificity=81.2%). When using Ct values
32.0 we found the best accuracy of multiplex PCR in detecting serotypes (Sensitivity
=90% and Specificity =84,7%). Conclusion: Our findings suggest that RT-PCR and
multiplex PCR techniques showed great potential to be used in evaluating the
vaccination impact. Further studies are needed to evaluate the cost-effectiveness of
using these technologies on a large scale.
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GARCIA, Weslley José Moreira. Uso de técnicas moleculares para determinação de Streptococcus pneumoniae e sorotipos colonizadores da nasofaringe na era pós-vacinal. 2013. 63 f. Dissertação (Mestrado em Medicina Tropical e Saúde Pública) - Universidade Federal de Goiás, Goiânia, 2013.